The potent cancer cell growth and tubulin assembly inhibitor combretastatin A-4 was originally isolated from the African tree Combretum caffrum (Combretaceae) circa 1985 and has been undergoing preclinical development. However, because of the very sparing aqueous solubility behavior of the phenol and its alkali metal salts, drug formulation attempts have given unsatisfactory results. The present invention represents a breakthrough in the continuing effort to synthesize practical water soluble prodrugs based on combretastatin A-4 and is a significant advance over the early efforts described in U.S. Pat. No. 5,561,122, supra.
In certain African Zulu traditional practices, the root bark of Combretum caffrum (C. salicyolium, combretaceae Family) is used as a charm for causing harm to an enemy. Combretastatin A-4, 1a, a Z-stilbene, isolated (Pettit et al., 1989, Isolation and structure of the strong cell-growth and tubulin inhibitor combretastatin A-4, Experimentia, 45, 209) from this tree has been shown to be a potent cancer cell growth inhibitor (EI-Zayat et al., 1993, In vitro evaluation of the antineoplastic activity of combretastatin A-4, a natural product from Combretum caffrum (arid shrub), Anti-Cancer Drugs, 4, 19); cancer anti-agent (Dark et al., 1997, combretastatin A-4, an agent that displays potent and selective toxicity towards tumour vasculature); and a tubulin assembly inhibitor (Lin et al., 1989, Antimitotic natural products combretastatin A-4 and combretastatin A-2: studies on the mechanism of their inhibition of the binding of colchicine to tubulin, Biochemistry, 28, 6984). Combretastatin a-4 is now in clinical development.
Although combretastatin A-4 has a phenol hydroxyl group and has demonstrated considerable promise as a unique anticancer agent, its development has been inhibited by its extremely poor solubility in water. A series of water-soluble derivatives have been recently reported (Brown et al., 1995, Synthesis of water-soluble sugar derivatives of combretastatin A-4, Journal of the Chemical Society, Perkin Transactions I, 577; Woods et al., 1995, The interaction with tubulin of a series of stilbenes based on combretastatin A-4, British Journal of Cancer, 71, 705; and, Bedford et al., 1996, Synthesis of Water-Soluble Prodrugs of the Cytotoxic Agent combretastatin A-4, Bioorganic & Medicinal Chemistry Letters, 6, 157) where the water-soluble phosphate sodium salt, 1h, (Pettit et al., 1995, Antineoplastic agents 322, Synthesis of combretastatin A-4 prodrugs, Anti-Cancer Drug Design, 10, 299; and, Pettit, U.S. Pat. No. 5,561,122) proved to be the most attractive. However, the phosphorylation sequence employing bis(2,2,2-trichloroethyl)phosphoro-chloridate, subsequent reduction (Zn, CH3CO2H), and isolation by ion-exchange chromatography was not well suited to producing prodrug 1h on a large scale and as such, posed a significant economic obstacle to its ultimate commercialization.
While prodrug options are steadily increasing and include poly(ethylene glycol) esters (Greenwald et al., 1996, Drug delivery systems: water soluble taxol 2′-poly(ethylene glycol) ester prodrugs-design and in vivo effectiveness, The Journal of Medicinal Chemistry, 39, 424); quaternary salts (Lackey et al., 1996, Water soluble inhibitors of topoisomerase I: quaternary salt derivatives of camptothecin, The Journal of Medicinal Chemistry, 39, 713), sulfonate salts (Heichman et al., 1995, Synthesis and cytotoxicity of water-soluble ambrosin prodrug candidates, The Journal of Medicinal Chemistry, 38, 3407); urethans (Izawa et al., 1995, Design and synthesis of an antitumor prodrug released by the reaction with sulfhydryl compounds, Bioorganic & Medicinal Chemistry Letters, 5, 593); biodegradable polymers (Gombotz et al., 1995, Biodegradable polymers for protein and peptide drug delivery, Bioconjugate Chemistry, 6, 332); and, a variety of other methods (Jungheim et al., 1994, Design of antitumor prodrugs: substrates for antibody targeted enzymes, Chemical Reviews, 94, 1553), the present disclosure is focused on the previously introduced sodium phosphate derivative 1h (which is equivalent to the sodium phosphate derivative 3d, produced by the methods set forth herein, see below) of combretastatin A-4. From evidence at hand phosphate 1h (as well as 3d) is presumed to be appropriately dephosphorylated by serum phosphatases and then transported intracellularly.
Furthermore, the phosphate prodrug approach has proved useful with substances as diverse as Etoposide (Saulnier et al., 1994, Synthesis of etoposide phosphate, BMY-40481: a water-soluble clinically active prodrug of etoposide, Bioorganic & Medicinal Chemistry Letters, 4, 2567); Taxol (Mamber et al., 1995, Tubulin polymerization by paclitaxel (Taxol) phosphate prodrugs after metabolic activation with alkaline phosphatase, The Journal of Pharmacology and Experimental Therapeutics, 374, 877; Ueda et al., 1995, Novel, water-soluble phosphate derivatives of 2′-ethoxycarbonylpaclitaxel as potential prodrugs of paclitaxel: synthesis and anti tumor evaluation, Bioorganic & Medicinal Chemistry Letters, 5, 247; and Ueda, et al., 1993, Novel water soluble phosphate prodrugs of Taxol possessing in vivo antitumor activity, Bioorganic & Medicinal Chemistry Letters, 3, 1761); and tyrosine-containing peptides (Chao et al., 1993, N,N-Diisopropyl-bis[2-(trimethylsily)ethyl]phosphoramidite, An attractive phosphorylating agent compatible with the Fmoc/t-butyl strategy for the synthesis of phosphotyrosine containing peptides, Tetrahedron Letters, 34, 3377). As shown herein below for prodrug 1h (also, 3d), phosphate ester salts are generally cleaved in vivo (Bundgaard (ed.), 1985, Design of prodrugs, pp. 1-92, Elsevier: New York) and are stable enough to usually allow formulation into solutions with practical shelf lives (Flynn et al., 1970, Factors influencing solvolysis of corticosteroid-21-phosphate esters, Journal of Pharmaceutical Sciences, 59, 1433). To improve the synthetic scaleup practicality for the sodium phosphate ester prodrug 1h (3d) of combretastatin A-4, three new phosphate esters have been synthesized which were easily transformed into the water-soluble prodrug 3d as is herein described.
The aggressive behavior of a broad spectrum of human cancer types such as breast carcinoma is related to the facility for rapid tumor angiogenesis. Neovascularization represents a very attractive target for new tumor antiangiogenesis-type anticancer drugs. Combretastatin A-4 1a was found to show potent antiangiogenesis activity and the derived sodium phosphate prodrug 1h (3d) is a powerful in vivo inhibitor of tumor vascularization. Currently prodrug 1h is in clinical development. A practical synthetic procedure for providing the pure drug in quantity for clinical trials has continued to be an important research objective. As described, the present invention is predicated upon the discovery of a considerably improved sequence utilizing in situ prepared dibenzyl chlorophosphite for phosphorylating phenol 1a, cleavage of the benzyl ester groups by trimethyliodosilane and treatment of the product with sodium methoxide to afford prodrug 1h (herein 3d) in good yield. However, the product was found to be accompanied by an isomeric substance that was first revealed by the appearance of some opalescence in aqueous solutions of prodrug 3d. The side product was believed to be the corresponding trans-stilbene 4c and was eliminated by extraction and fractional recrystallization. The purity of prodrug 3d was readily assigned by an ion-pair HPLC analysis using a phosphate buffer.
This further study was focused on confirming the structure of the persistent by-product (assumed to be 4c) by synthesis. In addition, the evaluation of combretastatin A-4 prodrugs was extended by preparing an extended series of phosphate metal and ammonium cation derivatives of the phosphoric acid precursor of prodrug 3d. Synthesis of the trans-stilbene phosphate 4c was also considered potentially useful for SAR purposes in view of the chemopreventative activities of certain trans-stilbene phenols such as resveratrol.
Combretastatin A-4 is essentially insoluble in water. This characteristic has significantly interfered with accomplishing the necessary formulations of pharmaceutical preparations of this compound for use in pre-clinical development. Hence, derivatives of the combretastatin A-4, 1a, 3′ phenol group were prepared for evaluation as possible water soluble prodrugs. As noted in U.S. Pat. No. 5,561,122, the sodium salt 1b, potassium salt 1c and hemisuccinic acid ester 1d derivatives of phenol 1a were essentially insoluble in water. Indeed, these substances regenerated combretastatin A-4 upon reaction with water. A series of other simple derivatives proved unsatisfactory in terms of water solubility or stability or both. The most soluble derivatives evaluated included the ammonium 1f, potassium 1g and sodium 1h phosphate salts where the latter two proved most stable and suitable. Both the sodium and other phosphate salt derivatives of combretastatin A-4 described herein were also found to exhibit the requisite biological properties necessary for a useful prodrug.
